A super-aging society has arrived, the number of people with osteoporosis has increased, and bone fractures resulting therefrom have come to constitute a serious issue of concern at a societal level. In particular, patients with femoral neck fractures and vertebral body fractures become bedridden, which causes significant deterioration of the quality of life thereof, and the social, medical, and economic burdens caused by care and hospital treatment have increased (Non-Patent Literatures 1 and 2).
It has also been discovered in recent years that osteoporosis is significantly associated with mortality in old age (Non Patent Literatures 3 and 4). Under such circumstances, prevention and treatment of osteoporosis have become critical objectives to be achieved.
Osteoporosis (i.e., a pathological condition where bone mass is reduced while the rate of the amount of the bone matrix to the amount of the mineralized bone matrix is held) is classified as primary osteoporosis or secondary osteoporosis. The former type is a pathological condition heretofore referred to as postmenopansal osteoporosis or senile osteoporosis. The latter type is a pathological condition caused by changes in bone metabolism resulting from other diseases, and such osteoporosis is classified based on the cause thereof, such as osteoporosis caused by endocrine, nutritional/metabolic, inflammatory, immobile, drug-induced, hematologic, congenital, or other diseases.
According to the above classification, endocrine causes include such as hyperparathyreosis, hyperthyreosis, hypogonadism, Cushing's syndrome, somatotropin deficiency, diabetes, Addison's disease, and calcitonin deficiency.
Nutritional/metabolic causes include such as chronic degenerative diseases, emaciation, serious liver diseases (primary biliary cirrhosis, in particular), gastric resection, scorbunts, malabsorption syndrome (including celiac disease), hypophosphatemia, chronic renal disease, hypercalciuria, hemochromatosis, amyloidosis, mast cell tumor, ingestion of excess sodium, insufficient calcium intake, and hypervitaminosis D or A. Inflammatory causes include such as articular rheumatism, particular bone disease (elevated bone resorption induced by proinflammatory cytokines), and sarcoidosis.
Immobility-related causes include such as systemic, bed rest, paralysis, local, and post-fracture causes. Drug-induced causes include such as with the use of steroids (steroids are extensively used for inflammatory diseases as immunosuppressive agents; examples of diseases treated with the use of steroids include collagen diseases, asthma, inflammatory bowel diseases, and in the case of organ transplantation, and bone loss is a serious side effect of such therapy), methotrexate, heparine, warfarin, anticonvulsant agents, lithium, and tamoxifen.
Blood-disease-induced causes include such as multiple myeloma, lymphoma, leukaemia, hemophilia, and chronic hemolytic diseases. Congenital causes include such as dysosteogenesis, Marfan's syndrome, Kleinfelter's syndrome, congenital erythropoetic porphyria, and cystic fibrosis.
Other disease-induced causes include such as with chronic obstructive lung diseases, hepatic failure, renal diseases, articular rheumatism, pregnancy, hyperoxemia, and HIV infection (Non-Patent Literature 5).
Among the above-mentioned diseases, bone diseases resulting from osteoarthritis, articular rheumatism, malignant tumors, or renal diseases are specifically regarded as bone diseases that impose serious influences at the societal level, in addition to primary osteoporosis. Osteoarthritis develops most often in locomotor regions. The number of patients afflicted therewith is said to be 10,000,000 in Japan, and it has been deduced that the number of patients will keep increasing as the aging of society advances. Advanced articular disorders are treated via artificial joint replacement; however, radical treatment of moderate or milder symptoms has not yet been reported (Non-Patent Literature 6).
Articular rheumatism is a chronic and progressive inflammatory disease characterized mainly by multiple arthritis. Articular synovial proliferation gradually causes infiltration of cartilage or bones in the vicinity thereof, and articular rheumatism often leads to destruction and deformation of joints. It has been reported that treatment with the use of an antirheumatic drug (methotrexate) cannot sufficiently inhibit the progress of joint destruction, and a biological agent targeting a tumor necrosis factor (TNF) α produces significant effects of inhibiting joint destruction. Thus, it is considered to be a revolutionary agent. However, increased incidence, as a side effect, of opportunistic infection, tuberculosis (extrapulmonary tuberculosis), Pneumocystispneumonia, or the like when using such agent is an issue of concern (Non-Patent Literature 7).
Major examples of bone diseases involved in malignant tumors include hypercalcemia and bone metastasis related to malignant tumors. Hypercalcemia causes loss of appetite and diuresis, and it causes dehydration and renal failure caused thereby. Bone metastasis is often observed in patients with breast cancer, prostate cancer, or lung cancer, in particular. While bone metastasis is hardly ever fatal by itself, it causes bone ache, pathologic fracture, neuroparalysis, or the like. It thus often significantly deteriorate patients' QOL, and bone metastasis control is a critical objective in clinical settings (Non-Patent Literature 8). These bone diseases related to malignant tumors are treated with the use of bisphosphonate preparations, although the problem of side effects has been pointed out.
Among bone diseases related to renal diseases, a pathological condition of bone damage caused by renal tissue damage is referred to as renal osteodystrophy. Bone disease experienced by kidney dialysis patients are mainly caused by secondary hyperparathyreosis. Because of the elevated PTH concentration caused by hyperparathyreosis and, for example, insufficient production of bone morphogenetic protein (BMP) 7, renal osteodystrophy advances. Dialysis patients often exhibit lowered reactivity of the bone with the parathyroid hormone (PTH). When the PTH concentration is chronically and significantly elevated, accordingly, fibrous ostitis (high bone turnover) develops. When the PTH concentration is maintained within a standard range, in contrast, bone aplasia (low bone turnover) develops.
When fibrous ostitis advances, collagen fibers are irregularly formed, such fibers are mineralized as non-crystalline calcium phosphate, and woven bone is then formed. This enhances bone formation, although the bone becomes easily fracturable. Basic treatment of fibrous ostitis involves inhibition of parathyroid hormone secretion, which mainly entails calcium ingestion and administration of active vitamin D.
When a patient has a chronic kidney disease (CKD) and receives dialysis treatment, in particular, various regulations, such as restrictions on food or water intake, are necessary. When secondary hyperparathyreosis advances, hypercalcemia also becomes an issue of concern. When prescribing active vitamin D, extreme caution, such as via the monitoring of renal functions (i.e., serum creatinine level) and serum calcium level, is always required.
Bone aplasia develops because of prolonged use and excessive administration of active vitamin D preparations or suppression of parathyroid hormone after parathyroidectomy (PTX). The rate of fractures associated with bone aplasia is higher than that associated with fibrous ostitis, and it induces hypercalcemia or mineralization of blood vessels or other soft tissues. Thus, adequate treatment techniques have been desired. A pathological condition of bone aplasia is low bone turnover in which bone resorption and bone formation are inhibited, and there is no established treatment technique at present (Non-Patent Literature 9).
Hyperphosphatemia or hypercalcemia caused by lowered capacity of the bone for phosphorus or calcium intake (low-turnover metabolic bone) or lowered storage capacity (high-turnover metabolic bone) is considered to be a cause of ectopic (vascular) mineralization. Cardiovascular complications account for 40% or more of the deaths of patients with chronic renal failures, and dialysis patients in particular, and arteriosclerosis involving vascular mineralization has drawn attention as a serious pathological condition. Treatment of mineralization of advanced lesions in patients with chronic renal failures remains difficult at present and the prognosis thereof is poor (Non-Patent Literature 10).
In addition to agents for treating primary osteoporosis, accordingly, development of agents that more effectively act on bone diseases resulting from osteoarthritis, articular rheumatism, malignant tumors, or renal disease and vascular mineralization resulting from bone diseases with reduced side effects has been desired.
It is considered that bone metabolism is regulated by the balance between osteoblast functions and osteoclast functions, and osteoporosis develops when the bone-destroying activity exceeds bone-building activity (Non-Patent Literature 11). In particular, secretion of the female hormone that assumes the role of protecting bones is lowered in postmenopausal women, a lowered capacity of osteoblasts for bone formation and the elevated bone resorption activity of osteoclasts are consequently observed, and it is highly likely that symptoms of osteoporosis would develop (Non-Patent Literatures 12 and 13).
In order to overcome such problems, estrogen preparations have been used; however, application thereof has been restricted due to the increased risk of thrombosis and breast cancer caused by the use of such preparations. It is also reported that use of a selective estrogen receptor modulator would increase the risk of deep vein thrombosis (Non-Patent Literature 14).
At present, calcitonin, bisphosphonate, and the like are used as agents that inhibit the bone resorption activity of osteoclasts. Calcitonin is known to bind to a calcitonin receptor expressed on the osteoclast surface to inactivate osteoclasts, and it is used for treatment of not only osteoporosis but also hypercalcemia, Paget's disease of bone, and the like in clinical settings.
However, no effects thereof on bone fracture inhibition have yet been found, and calcitonin receptor expression is reported to be down-regulated by calcitonin administration (Non-Patent Literatures 14 and 15). Bisphosphonate exhibits potent bone resorption inhibitory activity, and amino-containing bisphosphonates, such as andronate and risedronate, are major therapeutic agents for osteoporosis in Japan. Such bisphosphonate preparations inhibit farnesyl diphosphate synthase, block lipid protein prenylation, and induce inhibition of bone-resorption functions and osteoclast apoptosis (Non-Patent Literature 16).
However, the FDA warned of crises of severe skeletal, articular, or muscular pain in 2008 as problems of bisphosphonate preparations. In addition, side effects, such as jaw bone necrosis, caused by the prolonged use thereof (i.e., for 2 or 3 years or longer) after dental care have been reported (Non-Patent Literature 17).
An anti-RANK antibody has been expected as a novel osteoclastic inhibitor other than those described above. Further, application of the anti-RANK antibody as an inhibitor of articular destruction in the case of articular rheumatism or as a therapeutic agent for multiple myeloma has been expected, and clinical development thereof is in progress.
Based on a report to the effect that the RANKL/RANK pathway is important for the survival and maintenance of dendritic cells (Non-Patent Literature 18), or a report to the effect that lymph node dysplasia is caused in an RANK- or RANKL-deficient mouse (Non-Patent Literatures 19 and 20), the influence of an anti-RANK antibody preparation on the immune system has become an issue of concern.
In 2008, AMGEN reported that an increased rate of development of some infectious diseases was found through a clinical test of the anti-RANK antibody preparation (Denosumab). As a result of the clinical test of the anti-RANK antibody conducted in 2009, development of jaw bone necrosis was found to be a side effect, as in the cases of the bisphosphonate preparations.
Treatment via intermittent administration of PTH alone as an osteogenesis accelerator that activates osteoblasts has been conducted (Eli Lilly, teriparatide), but such agent is not different from other therapeutic agents, such as bisphosphonate preparations, in that activity of increasing cortical bone thickness is not very high compared with activity of increasing cancellous bone mass.
Accordingly, the effects thereof for bone fracture prevention are not considered to be very high. In relation to PTH, further, Asahi Kasei Pharma Corp. (Japan) has reported problems, such as side effects such as palpitation, tachycardia, and a lowering in blood pressure, and osteosarcoma observed in a long-term administration test to rats, unapproved continuous use thereof for 1.5 to 2 years or longer in Europe and the United States, and prohibited application thereof to cancer patients. Thus, it is impossible to use PTH for inhibition of cancer bone metastasis, treatment of cancer induced hypercalcemia (paraneoplastic humoral hypercalcemia or local osteolytic hypercalcemia caused by the parathyroid-hormone-related peptide produced by tumor cells), or other purposes.
Accordingly, development of agents that more effectively work for osteoporosis caused by the lowered capacity of osteoblasts for bone formation or elevated bone resorption activity of osteoclasts in postmenopausal women, hypercalcemia, Paget's disease of bone, inhibition of bone metastasis, inhibition of articular destruction associated with articular rheumatism, or multiple myeloma with reduced side effects has been awaited.
In addition thereto, osteohalisteresis andrachitis are known as bone diseases induced by selective inhibition of mineralization, unlike osteoporosis. A bone is formed by mineralization of a matrix layer comprising collagen or the like via hydroxyapatite deposition. Osteohalisteresis is a pathological condition in which such mineralization is blocked and osteoids increase, and it is referred to as rachitis if developed during childhood.
Symptoms include bone and joint pains, such as chiropodalgia, arthralgia, lumbago, and backache, which lead to gait impairment and to a state in which bone is easily fractured. In the case of children, developmental disorders, limb deformities such as bow-legs, pigeon breast deformity, or other symptoms are observed. Such symptoms are generally treated with the use of vitamin D, calcium preparations, and phosphorus preparations, in addition to alimentary therapy. If the level of dysfunction caused by a deformity is high, however, surgical operation is the only possible symptomatic treatment. Therefore, development of agents that are more effective on osteohalisteresis or rachitis has been awaited.
As described above, bone is tissue that is always regulated by the balance between osteoblast functions and osteoclast functions and remodeled. In order to achieve tough bone that is more resistant to fracture, accordingly, a mere increase in bone mass may not be sufficient.
In the case of hereditary diseases, such as osteopetrosis (Non-Patent Literature 21), Paget disease of bone (Non-Patent Literature 22) or Camurati-Engelmann's disease (CED) (Non-Patent Literatures 23 and 24), for example, it is known that the balance between bone formation and bone resorption becomes abnormal due to different causes, and bone strength is lowered even though bone mass is increased.
Examples of factors that determine bone strength from the viewpoint of mechanisms of materials include form-related factors, such as connectivity of cancellous bones, thickness of cortical bones, porosity, and cross-sectional moment, and qualitative factors, such as mineralization or bone fatigue, in addition to quantitative factors represented by bone density (Non-Patent Literature 25). Therefore, development of agents useful for improving bone strength, in addition to increasing bone mass, has been awaited for the purpose of treatment of primary osteoporosis and secondary osteoporosis.
In recent years, factors associated with the Wnt/LRP signal control mechanism have drawn attention as targets for drug discovery regarding a bone formation accelerator. Wnt is a secreted glycoprotein that has been lipid-modified by palmitic acid having a molecular weight of about 40,000, and 19 types thereof are considered to be present in mammalian animals. As Wnt receptors, 10 types of seven-transmembrane receptors (i.e., Frizzled receptors) and two types of single transmembrane receptors (i.e., LRP5/6 receptors) have been reported (Non-Patent Literature 26).
A region referred to as a cysteine-rich domain (CRD) containing conserved 10 cysteine residues is present in an extracellular region of the Frizzled receptor family molecule to which Wnt is considered to bind. The region from the cysteine residue located closest to the N-terminus to the cysteine residue located closest to the C-terminus of such 10 cysteine residues may be exclusively designated as a CRD (Non-Patent Literature 27), or a region comprising such 10 cysteine residues and sequences each located closer to the C- or N-terminus may be designated as a CRD (R&D systems). CRDs were reported to have homodimer structures based on crystal structural analysis using a CRD of mouse Frizzled 8 (Non-Patent Literature 28).
At least three types of Wnt signaling pathways are considered to exist: a canonical-Wnt signaling pathway; a non-canonical Wnt signaling pathway, which is a PCP (planar cell polarity) pathway mediated by a small G-binding protein; and a Ca2+ pathway mediated by a trimeric G protein. Bone-metabolism-related research on the canonlcal-Wnt signaling pathway is the most advanced, and Wnt is considered to promote bone formation (Non-Patent Literature 29). Therefore, regulation of functions of endogenous factors that inhibit this signaling pathway has been attempted in recent years for the purpose of application thereof to treatment of bone diseases.
Sclerostin was recognized as a BMP antagonist at first; however, it was reported to be a factor that would directly bind to LRP5/6 to inhibit the signaling pathway in research conducted later (Non-Patent Literature 30). A significant increase was observed in bone density in a Sclerostin-knockout mouse (Non-Patent Literature 31). At present, a Sclerostin-neutralizing antibody is undergoing phase II trials in Europe and the United States of America (AMG785, Amgen & UCB), and the future development thereof has drawn attention.
A DKK1 (Dickkopf-1)-neutralizing antibody that is known as another canonlcal-Wnt signal inhibitor was prepared, inhibition of lowered bone density was observed in an SCID mouse into which multiple myeloma (MM) cells had been transplanted (Non-Patent Literature 32), and clinical trials using a neutralizing antibody (BHQ880, Novartis) have been conducted.
sFRP (soluble frizzled-related protein) that is considered to be a Wnt decoy receptor and has high amino acid sequence homology to the Frizzled extracellular domain is considered to negatively regulate Wnt signals (Non-Patent Literature 33), and an increase in the amount of cancellous bone in the femur of an sFRP1 knockout mouse has been reported (Non-Patent Literature 34). Under such circumstances, research and development related to sFRP1 inhibitors have proceeded (Wyeth).
Frizzled 7 has been identified as a receptor that binds to a Wnt ligand and transmits signals thereof (Non-Patent Literatures 35 and 36). The amino acid sequence of the human Frizzled 7 extracellular cysteinerich domain (when a region from the cysteine residue located closest to the N-terminus to the cysteine residue located closest to the C-terminus of such 10 conserved cysteine residues is exclusively designated as a CRD) is completely identical to that of the mouse Frizzled 7 extracellular cysteine-rich domain (i.e., there is no difference between species). Involvement thereof with generation and differentiation of individual organisms (Non-Patent Literature 37) and involvement thereof with liver cell multiplication (Non-Patent Literature 38) have been reported.
Expression patterns of such molecules have been reported: an expression pattern localized in the crypt base of the mouse small intestine or large intestine (Non-Patent Literature 39); elevated expression levels in various cancer cells (Non-Patent Literature 40); expression in various tissues (the brain, eyeball, heart, kidney, liver, lung, or spermary) other than those of the spleen via expression analysis of adult mouse-derived tissues of (Non-Patent Literature 35); and expression in tissue (the lung or kidney) other than those of the brain and the liver via expression analysis of human fetal tissue and potent expression in the skeletal muscle and relatively potent expression in the heart, weak expression in the brain, the placenta, and the kidney; and no expression in the lung, the liver, the pancreas, the spleen, the thymic gland, the prostate, the testicle, the ovary, the small intestine, or the large intestine via expression analysis of adult human-derived tissue (Non-Patent Literature 41).
An extracellular cysteine-rich domain that is a soluble receptor of the Frizzled receptor is considered to bind to Wnt and inhibit functions thereof. It is reported by an in vitro experimentation system that a fusion product of the Frizzled 7 extracellular cysteine-rich domain (comprising a region from the cysteine residue located closest to the N-terminus to the cysteine residue located closest to the C-terminus of the conserved 10 cysteine residues and sequences each located closer to the C- or N-terminus) and Fc (R & D Systems) inhibits stabilization of cytoplasmic β-catenin by Wnt3a (Non-Patent Literature 42).
Since the expression level of Frizzled 7 is elevated in cancer cells, it has drawn attention as a target molecule for tumor treatment (Patent Literature 2, Non-Patent Literature 43). Regarding colon cancer cells into which a vector that expresses a Frizzled 7 extracellular domain has been introduced, for example, growth thereof was inhibited to a greater extent in a xenograft tumor cell transplantation model compared with colon cancer cells into which a control vector had been introduced (Non-Patent Literature 44). This suggests the possibility that Frizzled 7 would be a target of drug discovery for tumor treatment.
As described above, 10 types of Frizzled family molecules have been reported, and Frizzled 1 and Frizzled 2 have been reported as molecules having particularly high primary sequence homology with Frizzled 7 in the extracellular cysteine-rich domain (when a region from the cysteine residue located closest to the N-terminus to the cysteine residue located closest to the C-terminus of the conserved 10 cysteine residues is exclusively designated as a CRD; Non-Patent Literature 22).
The amino acid homologies of Frizzled 7 in the cysteine rich domain (when a region from the cysteine residue located closest to the N-terminus to the cysteine residue located closest to the C-terminus of the conserved 10 cysteine residues is exclusively designated as a CRD) of such molecule to Frizzled 1 and Frizzled 2 are 91% and 93% respectively in humans and mice. That is, such amino acid sequence homology is very high.
As with the case of Frizzled 7, Frizzled 1 and Frizzled 2 do not show differences between mouse-derived and human-derived amino acid sequences in the cysteine rich domain (when a region from the cysteine residue located closest to the N-terminus to the cysteine residue located closest to the C-terminus of the conserved 10 cysteine residues is exclusively designated as a CRD); i.e., such sequences are 100% consistent with each other.
As with Frizzled 7, it is reported that both Frizzled 1 and Frizzled 2 interact with Wnt and Frizzled 1 interacts with Wnt3a to protect the hippocampal neuron from being destroyed by amyloid β peptide (Non-Patent Literature 45).
In addition, regarding Frizzled 1 expression patterns, potent expression in the heart, the placenta, the lung, the kidney, the pancreas, the prostate, and the ovary observed via expression analysis of adult human derived tissue and potent expression in the lung and the kidney observed via expression analysis of fetus-derived tissue have been reported (Non-Patent Literature 41).
Since the expression levels of both Frizzled 1 and Frizzled 2 are elevated in the case of colon cancer or breast cancer, the correlation thereof with canceration is suggested, and they have drawn attention as target molecules for tumor treatment (Patent Literature 1, Non-Patent Literatures 46 and 47). Further, it was reported that Frizzled 1 would not cause any changes in the phenotype of the Frizzled 1 gene-disrupted mouse (Non-Patent Literature 48).
In super-aging society, bone diseases involved in osteoporosis, arthritis deformans, rheumatoid arthritis and malignant tumors and treatment of these bone diseases is increasingly considered as socially important issue, and medicaments for treating bone diseases are vigorously researched and developed.
Kakitani and co-workers have found that a protein containing an extracellular cysteine-rich domain derived from Frizzled 1, Frizzled 2 or Frizzled 7 specifically promotes an increase in bone mass and bone strength (Patent Literature 3). However, there still remains a need for more improvement of secretion activity in production cells (productivity) and bone mass-increasing activity.